A Year in the Lab: PFGE and Bradford Sample Collection

Tuesday 6th May 2014

This week began on Tuesday, as Monday was a bank holiday. I started the week off by incubating my newly made plugs in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.

Wednesday 7th May 2014

I began today by collecting the bacterial plugs from incubation, removing the buffer and enzyme then incubating the plugs in 1 x wash buffer for an hour with agitation. This was then removed and replaced with 0.5 x TAE buffer. While they were incubating in TAE I prepared and poured a 1% agarose gel.

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This gel was then loaded with the plugs, an appropriate PFGE ladder and my new Lambda ladder before running overnight using the following settings:

  • 20 hours
  • 1% agarose gel
  • 0.5x TAE
  • 14ºC
  • 40-100 switch intervals per second
  • 6 V/cm
  • 30 minute stain, 10 minute destain

I decided to run this gel using the same settings as my last most successful PFGE gel, in order to visulise whether this new batch of plugs contained a more appropriate amount of bacterial DNA alongside my new lambda ladder.

Thursday 8th May 2014

Once my PFGE gel had finished I stained it for 30 minutes, destaining for 10 minutes before viewing under UV. The resulting gel was fairly disappointing, while the new ladder appeared blurred my original PFGE ladder was very faint and the Escherichia coli bacterial plugs slightly on the dark side. The settings I have optimised for my original PFGE ladder and Escherichia coli bacterial plugs did not give appropriate banding patterns for my new Lambda ladder.

PFGE Gel

Unfortunately I don’t have enough time to run another gel again this week to confirm that my new ladder is not running correctly, so I will have to wait until my Bradford sample collection is complete.

Finally I collected my autoclaved Duran flasks, extendable rod and gloves to take home so that we could head straight off to Bradford in the morning.

Friday 9th May 2014

I began today fairly early, meeting my research supervisor and driving up to Bradford for water collection. After around 2 hours we reached Bradford, and headed towards Riddlesdon to explore the stretch of the river Aire into which the nearby waste water treatment plant (WWTP) disposed it’s processed effluent. As neither of us had been here to collect water before we spent around an hour and half finding the best way down to the river, taking multiple samples down stream from the WWTP and finally collecting a control sample from upstream. It was really good to get to explore a new river collection site, especially with my supervisor, as it opens up the potential for students to come back to this site for further research work.

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Once we were happy with all the samples taken we headed back to Lincoln and into the lab to start water processing. I began by filtering my collected water through filter membranes and applying these to MLSA plates, using the same method as before. These plates were then incubated overnight at 44ºC.

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Saturday 10th May 2014

In order to get all my Bradford water samples processed and stocked I decided to work over the weekend. I removed the incubated MLSA plates and inoculated single colonies from the plates into separate LB broths. These were then incubated overnight at 44ºC.

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Sunday 11th May 2014

Today started with removing the incubated LB broths and inoculating each into lactose peptone water (LPW) and tryptone water (TW) before incubating overnight at 44ºC. 10μl of each overnight sample was then spread plated onto mueller hinton agar, allowed to absorb before applying with antibiotic susceptibility discs. These plates were also incubated overnight at 44ºC.

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I then inoculated each overnight sample onto an individual nutrient agar slope, storing at room temperature to allow slow growth and access to single colonies. Finally, I glycerol stocked all the samples to ensure I have reserves of all isolated Bradford Escherichia coli samples. I pipetted 500μl of each overnight LB broth culture into separate tubes before adding 500μl of 10% glycerol LB broth. These were then carefully labelled and stored in the freezer.

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Next week…

Now I have all my Bradford water samples processed, Escherichia coli isolated and stored I will be moving onto plasmid extraction using the QIAcube. This will again allow me to see which Escherichia coli isolates carry plasmids, and should therefore be investigated through PCR for the presence of specific genes (Bla genes and CTX-M).

If I have time I would also like to carry on finalising PFGE settings with my new Lambda ladder. Once finished, hopefully I will be able to begin producing individual bacterial plugs for each Escherichia coli sample that demonstrated banding patterns when ran on standard gel electrophoresis. As I have only made 10 plugs at one time from only 3 samples I will need to set aside plenty of time to make plugs from all my Lincoln and Bradford samples!

A Year in the Lab…

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A Walk in the Life: Doddington Village Walk (Part 2)

Doddington is probably one of my favourite places to visit in Lincolnshire. I first visited Doddington Hall and gardens in December when the whole place was beautifully decorated for Christmas, however the gardens and surrounding woodland were looking fairly bare. Since then I have visited just as the weather was changing to try out the Doddington grounds circular walk as well as dropping by on Sundays to browse the delicious fresh produce in the farm shop. With the weather feeling very summery this week I decided to try out a slightly longer walk exploring the village of Doddington!

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Doddington Village Walk

This walk starts from the car park we used when visiting Doddington Hall and grounds, which is off the main road through Doddington, Lincoln, LN6 4RU. Parking is free, easy to find and only takes around 10 minutes by car to get there from the centre of Lincoln.

Off we go…

1. Begin in the main car park across from the hall and turn right onto the road, so that the church is on your left (If you get the chance, definitely explore the church of Saint Peter). Walk a short way until you see Kennel Lane on your right.

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2. Turn right onto Kennel Lane and follow this, through the houses and out along a grassy bridleway (Looking out across the fields you can see Lincoln cathedral in the distance). Follow this into woodland and continue on, past the ponds of the rendering works, until you reach a T-junction (Watch out for water fowl on the ponds! We saw some moorhens and coots swimming about when we visited).

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3. Turn right and follow this track around the barrier, along the edge of the works until you emerge onto the road. Continue right and follow the road for 2.4miles over two crossroads (We saw plenty of rabbits, squirrels and pheasants running around in the fields and across the roads when they were quiet).

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5. After the second crossroad, look for a public bridleway which leads right along the very edge of woodland just before you reach open fields. Turn right onto this bridleway (Look out for hedgerow birds darting back and forth, as well as butterflies and bees) and follow this track, with panoramic views over the Trent Valley, back to Doddington village.

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6. The track passes the start of the Fishpond Walk and back to the road (I would definitely recommend visiting the fish ponds nearby if you have the chance). Turn left and after a very short distance, the car park is on your right.

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This walk gives a great tour around the village of Doddington, is 4.9miles/7.9km in length and took around 2 hours to complete. I really enjoyed the first and last parts of this walk, however there is a fairly long section of road walking which isn’t something I like very much in a walk. Through out the walk we saw lots of public footpaths leading off our route, so perhaps with some exploring we could avoid the road almost completely.

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While walking past the rendering work ponds we saw loads of trees and shrubs covered in so much white pollen that it looked like it had been snowing! As we passed through some farm buildings we watched swallows dive in and out of the rafters from their nests, which was really great to see 🙂 It was good to be able to explore parts of Doddington I haven’t visited before, especially around the fish pond and the surrounding woodland. I would recommend this walk, however I think I would change it up slightly in the middle if I did it again.

A Walk in the Life…

A Bake in the Life: Malteser Bars

If you are a regular reader of my blog you may have been able to tell that I love to bake! However, when I’m super busy (Which seems like always at the moment!) I have to switch to less time consuming recipes which still mean that I can make delicious things without spending hours in the kitchen. One of my favourite, quick and hard to resist sweets to make when I am short on time is chocolate fridge cake/tiffin bars. I have wanted to try out this adaptation on traditional tiffin for ages, so with very little time and hungry friends to please I finally got round to making them 🙂

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Malteser Bars

You will need:

3½ oz/100g Butter

3½ oz/100g Golden Syrup

5 oz/150g Milk Chocolate

8 oz/225g Digestive Biscuits

6 oz/175g Maltesers (Chocolate Malted Balls)

10 oz/300g White Chocolate

Grating of Milk Chocolate

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Let’s get started!

  • Begin by greasing and lining a 19x29cm/7½x10½in tin, before setting aside.
  • In a large saucepan combine the butter, golden syrup and milk chocolate, placing over a low heat.
  • Stirring continuously, melt until smooth and glossy then remove from the heat.

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  • In a plastic food bag bash the digestive biscuits into fine crumbs.
  • In another bag smash the maltesers 3/4 times until a few are broken and some remain whole.

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  • Stir the biscuit crumbs and maltesers into the melted butter, golden syrup and milk chocolate until combined.
  • Spread evenly in your prepared tin and allow to cool for a few minutes.
  • Melt the white chocolate carefully in the microwave, or in a bowl over gently simmering water – ensuring that the bowl doesn’t touch the water.
  • Pour over the chocolate biscuit base and then sprinkle over the grated milk chocolate (If you like you can take a fork and swirl the two together to create a marbled effect).
  • Allow to chill in the fridge until set.

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  • You are all done! 🙂

The combination of crunchy and slightly chewy maltesers with the white chocolate in these bars is delicious 🙂 I love how simple this recipe is and, seeing as they take no time at all, how amazing the final malteser bars look! The crushed digestive biscuits soak up the chocolate and form a base, so when you cut it into bars you reveal the maltesers hiding inside.

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It only takes a few minutes to melt together chocolate, butter and syrup before adding in the dry ingredients and then it’s into the fridge to chill. Once you have the basic recipe down for fridge cake/tiffin bars they are really easy to adapt to your personal tastes or to what ever you have in the cupboard! I have made traditional Chocolate Tiffin before, as well Fifteens – both of which I would definitely recommend trying out.

A Bake in the Life…

A Year in the Lab: Modifying Bacterial Plugs and Plasmid Identification

Monday 28th April 2014

This week started with preparing my previously extracted Lincoln Escherichia coli collection plasmid DNA samples for running on gel electrophoresis. As I had a large amount of samples to run, I decided to spend today preparing all the samples for pipetting and then run them tomorrow. Extracted plasmid DNA from each sample was combined with loading buffer and pure water, then stored in the fridge. I also prepared a 1Kb ladder in the same way to use as a reference. I made enough to run alongside 8 gels, using 15 plasmid samples, as each gel tank and comb had 16 wells in total. Finally I prepared 1x TAE buffer, which would be used to make agarose gels, run the gel in and stain afterwards.

Tuesday 29th April 2014

I decided to run two gels at a time as I felt I would be able to prepare, pipette and run this amount without getting confused or making silly mistakes. I began by preparing and pouring 2 1% agarose gels before placing them into the tanks, filling them with 1x TAE buffer and loading them with the plasmid DNA I prepared yesterday. As I haven’t liquid pipetted into wells for a loooong time, this was fairly difficult the first few times! However, after 30 samples I felt much more confident.

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I ran both gels at 110V for 1 hour (As I was using small gel electrophoresis tanks), stained for 15 minutes and viewed under UV. I was very happy with my first two gels, as expected several samples demonstrated various different banding patterns while a few did not. I made a note of the samples which did demonstrate banding as this indicated that these samples contained plasmid DNA. This is interesting for my research as through PCR it can be determined whether this plasmid DNA contains specific genes (Bla genes and CTX-M) which code for the production of ESBL’s that play a direct role in antibiotic resistance to monobactams, penicillins and cephalosporins.

Wednesday 30th April 2014

Today began with starting new bacterial plug production, focusing on reducing the amount of DNA present in order to try and help resolve the problems seen last week. I inoculated one LB broth with a single colony from a sloped Escherichia coli sample from the Lincoln collection and another with the reference strain ATCC 25922. These broths were incubated, shaking at 37ºC for 4 hours until their OD reached 0.6-0.8. I chose to reduce the OD slightly from before in order to help reduce the amount of DNA present within the plug and therefore the darkness of the banding patterns. Each bacterial broth was then processed as before, centrifuging before combining with cell suspension buffer and agarose, pipetting into molds, incubating in lysozyme and then in proteinase K overnight at 50ºC.

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Once finished I then continued gel electrophoresis of pre prepared plasmid DNA samples. This was conducted in the same way and I was able to run 4 new gels throughout the day. As on Tuesday I was very happy with the gel images produced and I again noted down the samples which did demonstrate banding patterns.

Thursday 1st May 2014

Once the plugs had finished incubation in proteinase K, each plug was washed to remove residual lysozyme, proteinase K and lysed components. The washing procedure and times were kept the same as previously, and plugs were then stored in 1x wash buffer at 4ºC.

I then finished off gel electrophoresis of my last pre prepared plasmid DNA samples. This was conducted in the same way as before and I was very pleased with the gel images produced from my final 2 gels.

PFGE Gel 4

Friday 2nd May 2014

I decided to spend Friday printing off copies of all the gel images from the week, identifying plasmid DNA banding patterns as well as labeling ladders and Lincoln collection samples. This allowed me to produce a list of samples which I will be carrying on to investigate through PCR, due to the presence of plasmid DNA.

Next week…

As my new CHEF DNA Size Standard Lambda Ladder has arrived and my new batch of bacterial plugs are ready I can see whether the reduction in DNA has been successful and use the new ladder to begin approximating the smaller bands visible. I had also hoped to be starting on PCR, however my Taq Mastermix and 50bp ladder haven’t arrived yet so I will have to wait and keep planning until they get here!

I will also FINALLY be collecting water samples from Bradford next Friday – which is very exciting as I can’t wait to begin a comparable Bradford collection of Escherichia coli isolates. The journey up to Bradford should take around 2-3 hours, there and back, so it’s looking like it will be a long day and an even longer weekend…

A Year in the Lab…

A Walk in the Life: Ingham (Part 2)

It was a long time ago that I first visited the beautiful village of Ingham and ever since I have wanted to try out a second walk there. So, with the weather staying fairly sunny and bright we decided to head out. In case you haven’t seen my previous Ingham post, this walk comes from a series of walks in Lincoln I found online, many of which I have already tried out – particularly from the Lincolnshire Stepping Out series.

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Ingham 

The walk begins on the village green, opposite the village hall and old school buildings. We got there by car from the centre of Lincoln in around 15 minutes. There is plenty of free parking outside the village hall, and around the village green.

Off we go…

1. Leave the car park and walk towards the village green. Keep to the left with the village shop on your right. Turn left and walk along West End. Following West End as it bends to the left and then right, becoming Long Lane.

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2. Continue to follow Long Lane, looking out for hedgerow birds (Blue/Great tits, Chaffinches, Goldfinches and Blackbirds) until the road bends left. To complete the longer walk to the coates, follow the road right.

3. If doing the shorter walk, keep straight along the lane, past the houses and onto a track.

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4. As the track bends right follow the footpath on the left into the field (When we visited this field was full of beautiful yellow oil seed). Walk straight across the field aiming to the footpath sign in the hedge line.

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5. Turn right and follow the field edge to the corner (If you stop here and look back you can see the working power stations at Cottam and West Burton). Turn left over the bridge and continue straight on along the field edge (Look and listen for skylarks hoovering over the fields!).

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6. Cross the bridge and continue along the field edge (The sound of the trickling stream underneath was particularly lovely to hear). At the end of the field cross the bridge on the right and walk with the dyke on your left heading towards the sewage works. Head to the left of the sewage works, cross the dyke, and continue with the hedge on your right (When we visited there where loads of swallows diving across the fields, which was great to see!).

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7. At the end of the field turn left towards the village. This footpath will lead you back to the Village Hall and car park.

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This walk actually has two parts: a longer walk down and around the coates (9 km/5½miles), and a shorter walk through Ingham’s surrounding countryside (2½ km/1½miles). We decided to follow the shorter walk, however we did extend it slightly by continuing along Long Lane before heading left at the junction for the coates. This made the walk a little longer than 2½ km/1½miles and took us through fields full of beautiful yellow oil seed covered in bees and butterflies.

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I really enjoyed this walk as it allowed us to explore the quiet lanes in Ingham village as well as grassy tracks through the surrounding farmland. We saw lots of wildlife throughout the walk, including a variety of hedgerow birds, skylarks and swallows. I’m glad we decided to extend the shorter walk, as I think it would have been a little bit quick for us! However, this is easily done due to the many public footpaths and bridleways around the village. I would definitely recommend visiting Ingham and enjoying one or all of it’s beautiful walks! 🙂

A Walk in the Life…